Methods for the identification of inhibitors of histidinol-phosphate as antibiotics

ABSTRACT

The present inventors have discovered that histidinol-phosphatase is essential for fungal pathogenicity. Specifically, the inhibition of histidinol-phosphatase gene expression in fungi results in small, non-sporulating lesions and reduced pathogenicity. Thus, histidinol-phosphatase can be used as a target for the identification of antibiotics, preferably antifungals. Accordingly, the present invention provides methods for the identification of compounds that inhibit histidinol-phosphatase expression or activity. The methods of the invention are useful for the identification of antibiotics, preferably antifungals.

FIELD OF THE INVENTION

[0001] The invention relates generally to methods for the identificationof antibiotics, preferably antifungals that affect the biosynthesis ofL-histidine.

BACKGROUND OF THE INVENTION

[0002] Filamentous fungi are the causal agents responsible for manyserious pathogenic infections of plants and animals. Since fungi areeukaryotes, and thus more similar to their host organisms than, forexample bacteria, the treatment of infections by fungi poses specialrisks and challenges not encountered with other types of infections. Onesuch fungus is Magnaporthe grisea, the fungus that causes rice blastdisease. It is an organism that poses a significant threat to foodsupplies worldwide. Other examples of plant pathogens of economicimportance include the pathogens in the genera Agaricus, Alternaria,Anisogramma, Anthracoidea, Antrodia, Apiognomonia, Apiosporina,Armillaria, Ascochyta, Aspergillus, Bipolaris, Bjerkandera,Botryosphaeria, Botrytis, Ceratobasidium, Ceratocystis, Cercospora,Cercosporidium, Cerotelium, Cerrena, Chondrostereum, Chryphonectria,Chrysomyxa, Cladosporium, Claviceps, Cochliobolus, Coleosporium,Colletotrichium, Colletotrichum, Corticium, Corynespora, Cronartium,Cryphonectria, Cryptosphaeria, Cyathus, Cymadothea, Cytospora,Daedaleopsis, Diaporthe, Didymella, Diplocarpon, Diplodia,Discohainesia, Discula, Dothistroma, Drechslera, Echinodontium, Elsinoe,Endocronartium, Endothia, Entyloma, Epichloe, Ersiphe, Exobasidium,Exserohilum, Fomes, Fomitopsis, Fusarium, Gaeumannomyces, Ganoderma,Gibberella, Gloeocercospora, Gloeophyllum, Gloeoporus, Glomerella,Gnomoniella, Guignardia, Gymnosporangium, Helminthosporium,Herpotrichia, Heterobasidion, Hirschioporus, Hypodermella, Inonotus,Irpex, Kabatiella, Kabatina, Laetiporus, Laetisaria, Lasiodiplodia,Laxitextum, Leptographium, Leptosphaeria, Leptosphaerulina,Leucytospora, Linospora, Lophodermella, Lophodermium, Macrophomina,Magnaporthe, Marssonina, Melampsora, Melampsorella, Meria, Microdochium,Microsphaera, Monilinia, Monochaetia, Morchella, Mycosphaerella,Myrothecium, Nectria, Nigrospora, Ophiosphaerella, Ophiostoma,Penicillium, Perenniporia, Peridermium, Pestalotia, Phaeocryptopus,Phaeolus, Phakopsora, Phellinus, Phialophora, Phoma, Phomopsis,Phragmidium, Phyllachora, Phyllactinia, Phyllosticta, Phymatotrichopsis,Pleospora, Podosphaera, Pseudopeziza, Pseudoseptoria, Puccinia,Pucciniastrum, Pyricularia, Rhabdocline, Rhizoctonia, Rhizopus,Rhizosphaera, Rhynchosporium, Rhytisma, Schizophyllum, Schizopora,Scirrhia, Sclerotinia, Sclerotium, Scytinostroma, Septoria, Setosphaera,Sirococcus, Spaerotheca, Sphaeropsis, Sphaerotheca, Sporisorium,Stagonospora, Stemphylium, Stenocarpella, Stereum, Taphrina,Thielaviopsis, Tilletia, Trametes, Tranzschelia, Trichoderma, Tubakia,Typhula, Uncinula, Urocystis, Uromyces, Ustilago, Valsa, Venrturia,Verticillium, Xylaria, and others. Related organisms in theclassification, oornycetes, that include the genera Albugo, Aphanomyces,Bremia, Peronospora, Phytophthora, Plasmodiophora, Plasmopara,Pseudoperonospora, Pythium, Sclerophthora, and others are alsosignificant plant pathogens and are sometimes classified along with thetrue fungi. Human diseases that are caused by filamentous fungi includelife-threatening lung and disseminated diseases, often a result ofinfections by Aspergillus fumigatus. Other fungal diseases in animalsare caused by fungi in the genera, Fusarium, Blastomyces, Microsporum,Trichophyton, Epidermophyton, Candida, Histoplamsa, Pneumocystis,Cryptococcus, other Aspergilli, and others. The control of fungaldiseases in plants and animals is usually mediated by chemicals thatinhibit the growth, proliferation, and/or pathogenicity of the fungalorganisms. To date, there are less than twenty known modes-of-action forplant protection fungicides and human antifungal compounds.

[0003] A pathogenic organism has been defined as an organism thatcauses, or is capable of causing disease. Pathogenic organisms propagateon or in tissues and may obtain nutrients and other essential materialsfrom their hosts. A substantial amount of work concerning filamentousfungal pathogens has been performed with the human pathogen, Aspergillusfumigatus. Shibuya et al. (Shibuya, K., M. Takaoka, et al. (1999) MicrobPathog 27: 123-31 (PMID: 10455003)) have shown that the deletion ofeither of two suspected pathogenicity related genes encoding an alkalineprotease or a hydrophobin (rodlet) respectively, did not reducemortality of mice infected with these mutant strains. Smith et al.(Smith, J. M., C. M. Tang, et al. (1994) Infect Immun 62: 5247-54 (PMID:7960101)) showed similar results with alkaline protease and theribotoxin restrictocin; Aspergillus fumigatus strains mutated for eitherof these genes were fully pathogenic to mice. Reichard et al. (Reichard,U., M. Monod, et al. (1997) J Med Vet Mycol 35: 189-96 (PMID: 9229335))showed that deletion of the suspected pathogenicity gene encoding,aspergillopepsin (PEP) in Aspergillus fumigatus, had no effect onmortality in a guinea pig model system, and Aufauvre-Brown et al(Aufauvre-Brown, A., E. Mellado, et al. (1997) Fungal Genet Biol 21:141-52 (PMID: 9073488)) showed no effects of a chitin synthase mutationon pathogenicity. However, not all experiments produced negativeresults. Ergosterol is an important membrane component found in fungalorganisms. Pathnogenic fungi that lack key enzymes in this biochemicalpathway might be expected to be non-pathogenic since neither the plantnor animal hosts contain this particular sterol. Many antifungalcompounds that affect this biochemical pathway have been described(Onishi, J. C. and A. A. Patchett (1990a, b, c, d, and e) U.S. Pat. Nos.4,920,109; 4,920,111; 4,920,112; 4,920,113; and 4,921,844, Merck & Co.Inc. (Rahway N. J.)) and (Hewitt, H. G. (1998) Fungicides in CropProtection Cambridge, University Press). D'Enfert et al. (D'Enfert, C.,M. Diaquin, et al. (1996) Infect Immun 64: 4401-5 (PMID: 8926121))showed that an Aspergillus fumigatus strain mutated in an orotidine5′-phosphate decarboxylase gene was entirely non-pathogenic in mice, andBrown et al. (Brown, J. S., A. Aufauvre-Brown, et al (2000) MolMicrobiol 36: 1371-80 (PMID: 10931287)) observed a non-pathogenic resultwhen genes involved in the synthesis of para-aminobenzoic acid weremutated. Some specific target genes have been described as havingutility for the screening of inhibitors of plant pathogenic fungi. Bacotet al. (Baecot, K. O., D. B. Jordan, et al (2000) U.S. Pat. No.6,074,830, E. I. du Pont de Nemours & Company (Wilmington Del.))describe the use of 3,4-dihydroxy-2-butanone 4-phosphate synthase, andDavis et al. (Davis, G. E., G. D. Gustafson, et al (1999) U.S. Pat. No.5,976,848, Dow AgroSciences LLC (Indianapolis Ind.)) describe the use ofdihydroorotate dehydrogenase for potential screening purposes.

[0004] There are also a number of papers that report less clear results,showing neither full pathogenicity nor non-pathogenicity of mutants.Hensel et al. (Hensel, M., H. N. Arxst, Jr., et al. (1998) Mol Gen Genet258: 553-7 (PMID: 9669338)) showed only moderate effects of the deletionof the area transcriptional activator on the pathogenicity ofAspergillus fumigatus.

[0005] Therefore, it is not currently possible to determine whichspecific growth materials may be readily obtained by a pathogen from itshost, and which materials may not. We have found that Magnaporthe griseathat cannot synthesize their own L-histidine have reduced pathogenicityon their host organism. The M. grisea HISPI enzyme has greatestsimilarity to Schizosaccharomyces pombe His9, as well as some similarityto Saccharomyces cerevisiae His2p and His9. These genes encode adistantly related family of Histidinol Phosphate Phosphatases (HolPase),which catalyzes the dephosphorylation of Histidinol Phosphate toHistidinol. This family includes the HolPase encoded by the HisJ (orytvP) gene found in Bacillus subtilis. Knock-out of HisJ has yielded anauxotrophic mutant, unable to grow without Histidine supplementation (leCoq et al. (1999) J Bacteriol 181: 3277-3280 (PMID: 10322033)). Noreferences were found where SpHis2, ScHis2p or ScHis9 are known targetsfor anti-fungal/fungicide development. However, S. cerevisiae mutantscontaining knock-outs in the His1-His7 genes have been shown to beunable to grow in elevated levels of Cu, Co, or Ni at near-neutral pH(Pearce and Sherman (1999) J Bacteriol 181: 4774-4779 (PMID: 10438744)).

[0006] To date there do not appear to be any publications demonstratingan anti-pathogenic effect of the knock-out, over-expression, antisenseexpression, or inhibition of the genes or gene products involved inL-histidine biosynthesis in filamentous fungi. Thus, it has not beenshown that the de novo biosynthesis of L-histidine is essential forfungal pathogenicity. And, thus, it would be desirable to determine theutility of the enzymes involved in L-histidine biosynthesis forevaluating antibiotic compounds, especially fungicides. If a fungalbiochemical pathway or specific gene product in that pathway is shown tobe required for fungal pathogenicity, various formats of in vitro and invivo screening assays may be put in place to discover classes ofchemical compounds that react with the validated target gene, geneproduct, or biochemical pathway, and are thus candidates for antifungal,biocide, and biostatic materials.

SUMMARY OF THE INVENTION

[0007] Surprisingly, the present inventors have discovered that in vivodisruption of the gene encoding histidinol-phosphatase in Magnaporthegrisea prevents or inhibits the pathogenicity of the fungus. Thus, thepresent inventors have discovered that histidinol-phosphatase isessential for normal rice blast pathogenicity, and can be used as atarget for the identification of antibiotics, preferably fungicides.Accordingly, the present invention provides methods for theidentification of compounds that inhibit histidinol-phosphataseexpression or activity. The methods of the invention are usefu for theidentification of antibiotics, preferably fungicides.

BRIEF DESCRIPTION OF THE FIGURES

[0008]FIG. 1 shows the reaction performed by histidinol-phosphatase(HISP1) reaction. The Substrates/Products are L-histidinol phosphate andH₂O and the Products/Substrates are L-histidinol and orthophosphate. Thefunction of the histidinol-phosphatase enzyme is the interconversion ofL-histidinol phosphate and H₂O to L-histidinol and orthophosphate. Thisreaction is part of the L-histidine biosynthesis pathway.

[0009]FIG. 2 shows a digital image showing the effect of HISP1 genedisruption on Magnaporthe grisea pathogenicity using whole plantinfection assays. Rice variety CO39 was inoculated with wild-type andthe transposon insertion strains, KO1-1 and KO1-3. Leaf segments wereimaged at five days post-inoculation.

[0010]FIG. 3. Verification of Gene Function by Analysis of NutritionalRequirements. Wild-type and transposon insertion strains, KO1-1 andKO1-3, were grown in (A) minimal media and (B) minimal media with theaddition of L-histidine, respectively. The x-axis shows time in days andthe y-axis shows turbidity measured at 490 nanometers and 750nanometers. The symbols represent wildtype (—♦—), transposon strainKO1-1 (—

—) and transposon strain KO1-3 (—▴—).

DETAILED DESCRIPTION OF THE INVENTION

[0011] Unless otherwise indicated, the following terms are intended tohave the following meanings in interpreting the present invention.

[0012] The term “active against” in the context of compounds, agents, orcompositions having antibiotic activity indicates that the compoundexerts an effect on a particular target or targets which is deleteriousto the in vitro and/or in vivo growth of an organism having that targetor targets. In particular, a compound active against a gene exerts anaction on a target which affects an expression product of that gene.This does not necessarily mean that the compound acts directly on theexpression product of the gene, but instead indicates that the compoundaffects the expression product in a deleterious manner. Thus, the directtarget of the compound may be, for example, at an upstream componentwhich reduces transcription from the gene, resulting in a lower level ofexpression. Likewise, the compound may affect the level of translationof a polypeptide expression product, or may act on a downstreamcomponent of a biochemical pathway in which the expression product ofthe gene has a major biological role. Consequently, such a compound canbe said to be active against the gene, against the gene product, oragainst the related component either upstream or downstream of that geneor expression product. While the term “active against” encompasses abroad range of potential activities, it also implies some degree ofspecificity of target. Therefore, for example, a general protease is not“active against” a particular gene which produces a polypeptide product.In contrast, a compound which inhibits a particular enzyme is activeagainst that enzyme and against the gene which codes for that enzyme.

[0013] As used herein, the term “allele” refers to any of thealternative forms of a gene that may occur at a given locus.

[0014] The term “antibiotic” refers to any substance or compound thatwhen contacted with a living cell, organism, virus, or other entitycapable of replication, results in a reduction of growth, viability, orpathogenicity of that entity.

[0015] The term “binding” refers to a non-covalent or a covalentinteraction, preferably non-covalent, that holds two molecules together.For example, two such molecules could be an enzyme and an inhibitor ofthat enzyme. Non-covalent interactions include hydrogen bonding, ionicinteractions among charged groups, van der Waals interactions and.hydrophobic interactions among nonpolar groups. One or more of theseinteractions can mediate the binding of two molecules to each other.

[0016] The term “biochemical pathway” or “pathway” refers to a connectedseries of biochemical reactions normally occurring in a cell, or morebroadly a cellular event such as cellular division or DNA replication.Typically, the steps in such a biochemical pathway act in a coordinatedfashion to produce a specific product or products or to produce someother particular biochemical action. Such a biochemical pathway requiresthe expression product of a gene if the absence of that expressionproduct either directly or indirectly prevents the completion of one ormore steps in that pathway, thereby preventing or significantly reducingthe production of one or more normal products or effects of thatpathway. Thus, an agent specifically inhibits such a biochemical pathwayrequiring the expression product of a particular gene if the presence ofthe agent stops or substantially reduces the completion of the series ofsteps in that pathway. Such an agent, may, but does not necessarily, actdirectly on the expression product of that particular gene.

[0017] As used herein, the term “cDNA” means complementarydeoxyribonucleic acid.

[0018] As used herein, the term “CoA” means coenzyme A.

[0019] As used herein, the term “conditional lethal” refers to amutation permitting growth and/or survival only under special growth orenvironmental conditions.

[0020] As used herein, the term “cosmid” refers to a hybrid vector, usedin gene cloning, that includes a cos site (from the lambdabacteriophage). It also contains drug resistance marker genes and otherplasmid genes. Cosmids are especially suitable for cloning large genesor multigene fragments.

[0021] As used herein, the term “dominant allele” refers to a dominantmutant allele in which a discernable mutant phenotype can be detectedwhen this mutation is present in an organism that also contains a wildtype (non-mutant), recessive allele, or other dominant allele.

[0022] As used herein, the term “DNA” means deoxyribonucleic acid.

[0023] As used herein, the term “ELISA” means enzyme-linkedimmunosorbent assay.

[0024] “Fungi” (singular: fungus) refers to whole fungi, fungal organsand tissues (e.g., asci, hyphae, pseudohyphae, rhizoid, sclerotia,sterigmata, spores, sporodochia, sporangia, synnemata, conidia,ascostroma, cleistothecia, mycelia, perithecia, basidia and the like),spores, fungal cells and the progeny thereof. Fungi are a group oforganisms (about 50,000 known species), including, but not limited to,mushrooms, mildews, moulds, yeasts, etc., comprising the kingdom Fungi.They can either exist as single cells or make up a multicellular bodycalled a mycelium, which consists of filaments known as hyphae. Mostfungal cells are multinucleate and have cell walls, composed chiefly ofchitin. Fungi exist primarily in damp situations on land and, because ofthe absence of chlorophyll and thus the inability to manufacture theirown food by photosynthesis, are either parasites on other organisms orsaprotrophs feeding on dead organic matter. The principal criteria usedin classification are the nature of the spores produced and the presenceor absence of cross walls within the hyphae. Fungi are distributedworldwide in terrestrial, freshwater, and marine habitats. Some live inthe soil. Many pathogenic fungi cause disease in animals and man or inplants, while some saprotrophs are destructive to timber, textiles, andother materials. Some fungi form associations with other organisms, mostnotably with algae to form lichens.

[0025] As used herein, the term “fungicide”, “antiftngal”, or“antimycotic” refers to an antibiotic substance or compound that killsor suppresses the growth, viability, or pathogenicity of at least onefungus, fungal cell, fungal tissue or spore.

[0026] In the context of this disclosure, “gene” should be understood torefer to a unit of heredity. Each gene is composed of a linear chain ofdeoxyribonucleotides which can be referred to by the sequence ofnucleotides forming the chain. Thus, “sequence” is used to indicate boththe ordered listing of the nucleotides which form the chain, and thechain, itself, which has that sequence of nucleotides. (“Sequence” isused in the similar way in referring to RNA chains, linear chains madeof ribonucleotides.) The gene may include regulatory and controlsequences, sequences which can be transcribed into an RNA molecule, andmay contain sequences with unknown function. The majority of the RNAtranscription products are messenger RNAs (mRNAs), which includesequences which are translated into polypeptides and may includesequences which are not translated. It should be recognized that smalldifferences in nucleotide sequence for the same gene can exist betweendifferent fungal strains, or even within a particular fungal strain,without altering the identity of the gene.

[0027] As used in this disclosure, the terms “growth” or “cell growth”of an organism refers to an increase in mass, density, or number ofcells of said organism. Some common methods for the measurement ofgrowth include the determination of the optical density of a cellsuspension, the counting of the number of cells in a fixed volume, thecounting of the number of cells by measurement of cell division, themeasurement of cellular mass or cellular volume, and the like.

[0028] As used in this disclosure, the term “growth conditionalphenotype” indicates that a fungal strain having such a phenotypeexhibits a significantly greater difference in growth rates in responseto a change in one or more of the culture parameters than an otherwisesimilar strain not having a growth conditional phenotype. Typically, agrowth conditional phenotype is described with respect to a singlegrowth culture parameter, such as temperature. Thus, a temperature (orheat-sensitive) mutant (i.e., a fungal strain having a heat-sensitivephenotype) exhibits significantly different growth, and preferably nogrowth, under non-permissive temperature conditions as compared togrowth under permissive conditions. In addition, such mutants preferablyalso show intermediate growth rates at intermediate, or semi-permissive,temperatures. Similar responses also result from the appropriate growthchanges for other types of growth conditional phenotypes.

[0029] As used herein, the term “H₂O” means water.

[0030] As used herein, the term “heterologous HISP1 gene” means a gene,not derived from Magnaporthe grisea, and having: at least 50% sequenceidentity, preferably 60%, 70%, 80%, 90%, 95%, 99% sequence identity andeach integer unit of sequence identity from 50-100% in ascending orderto SEQ ID NO: 1 or SEQ ID NO: 2; or at least 10% of the activity of aMagnaporthe grisea histidinol-phosphatase, preferably 25%, 50%, 75%,90%, 95%, 99% and each integer unit of activity from 10-100% inascending order.

[0031] As used herein, the term “HISP1” means a gene encodinghistidinol-phosphatase activity, referring to an enzyme that catalysesthe interconversion of L-histidinol phosphate and H₂O with L-histidinoland orthophosphate, and may also be used to refer to the gene product.

[0032] As used herein, the term “His-Tag” refers to an encodedpolypeptide consisting of multiple consecutive histidine amino acids.

[0033] As used herein, the terms “histidinol-phosphatase” (EC 3.1.3.15)and “histidinol-phosphatase polypeptide” are synonymous with “the HISP1gene product” and refer to an enzyme that catalyses the interconversionof L-histidinol phosphate and H₂O with L-histidinol and orthophosphate.

[0034] As used herein, the term “HPLC” means high pressure liquidchromatography.

[0035] As used herein, the terms “hph”, “hygromycin Bphosphotransferase”, and “hygromycin resistance gene” refer to the E.coli hygromycin phosphotransferase gene or gene product.

[0036] As used herein, the term “hygromycin B” refers to anaminoglycosidic antibiotic, used for selection and maintenance ofeukaryotic cells containing the E. coli hygromycin resistance gene.

[0037] “Hypersensitive” refers to a phenotype in which cells are moresensitive to antibiotic compounds than are wild-type cells of similar oridentical genetic background.

[0038] “Hyposensitive” refers to a phenotype in which cells are lesssensitive to antibiotic compounds than are wild-type cells of similar oridentical genetic background.

[0039] As used herein, the term “imperfect state” refers to aclassification of a fungal organism having no demonstrable sexual lifestage.

[0040] The term “inhibitor”, as used herein, refers to a chemicalsubstance that inactivates the enzymatic activity ofhistidinol-phosphatase or substantially reduces the level of enzymaticactivity, wherein “substantially” means a reduction at least as great asthe standard deviation for a measurement, preferably a reduction by 50%,more preferably a reduction of at least one magnitude, i.e. to 10%. Theinhibitor may function by interacting directly with the enzyme, acofactor of the enzyme, the substrate of the enzyme, or any combinationthereof.

[0041] A polynucleotide may be “introduced” into a fungal cell by anymeans known to those of skill in the art, including transfection,transformation or transduction, transposable element, electroporation,particle bombardment, infection and the like. The introducedpolynucleotide may be maintained in the cell stably if it isincorporated into a non-chromosomal autonomous replicon or integratedinto the fungal chromosome. Alternatively, the introduced polynucleotidemay be present on an extra-chromosomal non-replicating vector and betransiently expressed or transiently active.

[0042] As used herein, the term “knockout” or “gene disruption” refersto the creation of organisms carrying a null mutation (a mutation inwhich there is no active gene product), a partial null mutation ormutations, or an alteration or alterations in gene regulation byinterrupting a DNA sequence through insertion of a foreign piece of DNA.Usually the foreign DNA encodes a selectable marker.

[0043] As used herein, the term “LB agar” means Luria's Broth agar.

[0044] The term “method of screening” means that the method is suitable,and is typically used, for testing for a particular property or effectin a large number of compounds. Typically, more than one compound istested simultaneously (as in a 96-well microtiter plate), and preferablysignificant portions of the procedure can be automated. “Method ofscreening” also refers to the determination of a set of differentproperties or effects of one compound simultaneously.

[0045] As used herein, the term “mRNA” means messenger ribonucleic acid.

[0046] As used herein, the term “mutant form” of a gene refers to a genewhich has been altered, either naturally or artificially, changing thebase sequence of the gene. The change in the base sequence may be ofseveral different types, including changes of one or more bases fordifferent bases, deletions, and/or insertions, such as by a transposon.By contrast, a normal form of a gene (wild type) is a form commonlyfound in natural populations of an organism. Commonly a single form of agene will predominate in natural populations. In general, such a gene issuitable as a normal form of a gene, however, other forms which providesimilar functional characteristics may also be used as a normal gene. Inparticular, a normal form of a gene does not confer a growth conditionalphenotype on the strain having that gene, while a mutant form of a genesuitable for use in these methods does provide such a growth conditionalphenotype.

[0047] As used herein, the term “Ni” refers to nickel.

[0048] As used herein, the term “Ni-NTA” refers to nickel sepharose.

[0049] As used herein, a “normal” form of a gene (wild type) is a formcommonly found in natural populations of an organism. Commonly a singleform of a gene will predominate in natural populations. In general, sucha gene is suitable as a normal form of a gene, however, other formswhich provide similar functional characteristics may also be used as anormal gene. In particular, a normal form of a gene does not confer agrowth conditional phenotype on the strain having that gene, while amutant form of a gene suitable for use in these methods does providesuch a growth conditional phenotype.

[0050] As used herein, the term “one form” of a gene is synonymous withthe term “gene”, and a “different form” of a gene refers to a gene thathas greater than 49% sequence identity and less than 100% sequenceidentity with said first form.

[0051] As used herein, the term “pathogenicity” refers to a capabilityof causing disease. The term is applied to parasitic microorganisms inrelation to their hosts.

[0052] As used herein, the term “PCR” means polymerase chain reaction.

[0053] The “percent (%) sequence identity” between two polynucleotide ortwo polypeptide sequences is determined according to the either theBLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W.Gish, et al. (1990) J Mol Biol 215: 403-10 (PMID: 2231712)) at theNational Center for Biotechnology or using Smith Waterman Alignment(Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147: 195-7 (PMID:7265238)) as incorporated into GeneMatcher Plus™. It is understood thatfor the purposes of determining sequence identity when comparing a DNAsequence to an RNA sequence, a thymine nucleotide is equivalent to auracil nucleotide.

[0054] By “polypeptide” is meant a chain of at least two amino acidsjoined by peptide bonds. The chain may be linear, branched, circular orcombinations thereof. Preferably, polypeptides are from about 10 toabout 1000 amino acids in length, more preferably 10-50 amino acids inlength. The polypeptides may contain amino acid analogs and othermodifications, including, but not limited to glycosylated orphosphorylated residues.

[0055] As used herein, the term “proliferation” is synonymous to theterm “growth”.

[0056] As used herein, the term “reverse transcriptase-PCR” meansreverse transcription-polymerase chain reaction.

[0057] As used herein, the term “RNA” means ribonucleic acid.

[0058] As used herein, “semi-permissive conditions” are conditions inwhich the relevant culture parameter for a particular growth conditionalphenotype is intermediate between permissive conditions andnon-permissive conditions. Consequently, in semi-permissive conditionsan organism having a growth conditional phenotype will exhibit growthrates intermediate between those shown in permissive conditions andnon-permissive conditions. In general, such intermediate growth rate maybe due to a mutant cellular component which is partially functionalunder semi-permissive conditions, essentially fully functional underpermissive conditions, and is non-functional or has very low functionunder non-permissive conditions, where the level of function of thatcomponent is related to the growth rate of the organism. An intermediategrowth rate may also be a result of a nutrient substance or substancesthat are present in amounts not sufficient for optimal growth rates tobe achieved.

[0059] “Sensitivity phenotype” refers to a phenotype that exhibitseither hypersensitivity or hyposensitivity.

[0060] The term “specific binding” refers to an interaction betweenhistidinol-phosphatase and a molecule or compound, wherein theinteraction is dependent upon the primary amino acid sequence and/or theconformation of histidinol-phosphatase.

[0061] As used herein, the term “TLC” means thin layer chromatography.

[0062] “Transform”, as used herein, refers to the introduction of apolynucleotide (single or double stranded DNA, RNA, or a combinationthereof) into a living cell by any means. Transformation may beaccomplished by a variety of methods, including, but not limited to,electroporation, polyethylene glycol mediated uptake, particlebombardment, agrotransformation, and the like. This process may resultin transient or stable expression of the transformed polynucleotide. By“stably transformed” is meant that the sequence of interest isintegrated into a replicon in the cell, such as a chromosome or episome.Transformed cells encompass not only the end product of a transformationprocess, but also the progeny thereof which retain the polynucleotide ofinterest.

[0063] For the purposes of the invention, “transgenic” refers to anycell, spore, tissue or part, that contains all or part of at least onerecombinant polynucleotide. In many cases, all or part of therecombinant polynucleotide is stably integrated into a chromosome orstable extra-chromosomal element, so that it is passed on to successivegenerations.

[0064] As used herein, the term “transposase” refers to an enzyme thatcatalyzes transposition. Preferred transposons are described in WO00/55346, PCT/U.S.00/07317, and U.S. Ser. No. 09/658,859.

[0065] As used herein, the term “transposition” refers to a complexgenetic rearrangement process involving the movement or copying of apolynucleotide (transposon) from one location and insertion intoanother, often within or between a genome or genomes, or DNA constructssuch as plasmids, bacmids, and cosmids.

[0066] As used herein, the term “transposon” (also known as a“transposable element”, “transposable genetic element”, “mobileelement”, or “jumping gene”) refers to a mobile DNA element such asthose, for example, described in WO 00/55346, PCT/U.S.00/07317, and.U.S. Ser. No. 09/658,859. Transposons can disrupt gene expression orcause deletions and inversions, and hence affect both the genotype andphenotype of the organisms concerned. The mobility of transposableelements has long been used in genetic manipulation, to introduce genesor other information into the genome of certain model systems.

[0067] As used herein, the term “Tween 20” means sorbitanmono-9-octadecenoate poly(oxy-1,1-ethanediyl).

[0068] As used in this disclosure, the term “viability” of an organismrefers to the ability of an organism to demonstrate growth underconditions appropriate for said organism, or to demonstrate an activecellular function. Some examples of active cellular functions includerespiration as measured by gas evolution, secretion of proteins and/orother compounds, dye exclusion, mobility, dye oxidation, dye reduction,pigment production, changes in medium acidity, and the like.

[0069] The present inventors have discovered that disruption of theHISPI gene and/or gene product inhibits the pathogenicity of Magnaporthegrisea. Thus, the inventors are the first to demonstrate thathistidinol-phosphatase is a target for antibiotics, preferablyantifungals.

[0070] Accordingly, the invention provides methods for identifyingcompounds that inhibit HISP1 gene expression or biological activity ofits gene product(s). Such methods include ligand binding assays, assaysfor enzyme activity, cell-based assays, and assays for HISP1 geneexpression. Any compound that is a ligand for histidinol-phosphatase mayhave antibiotic activity. For the purposes of the invention, “ligand”refers to a molecule that will bind to a site on a polypeptide. Thecompounds identified by the methods of the invention are useful asantibiotics.

[0071] Thus, in one embodiment, the invention provides a method foridentifying a test compound as a candidate for an antibiotic,comprising:

[0072] a) contacting a histidinol-phosphatase polypeptide with a testcompound; and

[0073] b) detecting the presence or absence of binding between said testcompound and said histidinol-phosphatase polypeptide;

[0074] wherein binding indicates that said test compound is a candidatefor an antibiotic.

[0075] The histidinol-phosphatase protein may have the amino acidsequence of a naturally occurring histidinol-phosphatase found in afungus, animal, plant, or microorganism, or may have an amino acidsequence derived from a naturally occurring sequence. Preferably thehistidinol-phosphatase is a fungal histidinol-phosphatase. The cDNA (SEQID NO: 1) encoding the histidinol-phosphatase protein, the genomic DNA(SEQ ID NO: 2) encoding the M. grisea protein, and the polypeptide (SEQID NO: 3) can be found herein.

[0076] In one aspect, the invention also provides for a polypeptideconsisting essentially of SEQ ID NO: 3. For the purposes of theinvention, a polypeptide consisting essentially of SEQ ID NO: 3 has atleast 80% sequence identity with SEQ ID NO: 3 and catalyses theinterconversion of L-histidinol phosphate and H₂O with L-histidinol andorthophosphate with at least 10% of the activity of SEQ ID NO: 3.Preferably, the polypeptide consisting essentially of SEQ ID NO: 3 hasat least 85% sequence identity with SEQ ID NO: 3, more preferably thesequence identity is at least 90%, most preferably the sequence identityis at least 95% or 97 or 99%, or any integer from 80-100% sequenceidentity in ascending order. And, preferably, the polypeptide consistingessentially of SEQ ID NO: 3 has at least 25%, at least 50%, at least 75%or at least 90% of the activity of M. grisea histidinol-phosphatase, orany integer from 60-100% activity in ascending order.

[0077] By “fungal histidinol-phosphatase” is meant an enzyme that can befound in at least one fungus, and which catalyzes the interconversion ofL-histidinol phosphate and H₂O with L-histidinol and orthophosphate. Thehistidinol-phosphatase may be from any of the fungi, includingascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

[0078] In one embodiment, the histidinol-phosphatase is a Magnaporthehistidinol-phosphatase. Magnaporthe species include, but are not limitedto, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea andMagnaporthe poae and the imperfect states of Magnaporthe in the genusPyricularia. Preferably, the Magnaporthe histidinol-phosphatase is fromMagnaporthe grisea.

[0079] In various embodiments, the histidinol-phosphatase can be fromPowdery Scab (Spogngospora subterranea), Grey Mould (Botrytis cinerea),White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum),Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot(Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), HoneyFungus (Armillaria gallica), Root rot (Armillaria luteobubalina),Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus(Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena),Apple-rotting Fungus (Penicillium expansum), Clubroot Disease(Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Rootpathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomycesgraminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromycesappendiculatus), Northern Leaf Spot (Cochliobolus carbonum), MiloDisease (Periconia circinata), Southern Corn Blight (Cochliobolusheterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe(Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), WheatHead Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusariumculmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black StemRust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), and thelike.

[0080] Fragments of a histidinol-phosphatase polypeptide may be used inthe methods of the invention, preferably if the fragments include anintact or nearly intact epitope that occurs on the biologically activewildtype histidinol-phosphatase. The fragments comprise at least 10consecutive amino acids of a histidinol-phosphatase. Preferably, thefragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, or at least 330 consecutiveamino acids residues of a histidinol-phosphatase. In one embodiment, thefragment is from a Magnaporthe histidinol-phosphatase. Preferably, thefragment contains an amino acid sequence conserved among fungalhistidinol-phosphatases.

[0081] Polypeptides having at least 50% sequence identity with a fungalhistidinol-phosphatase are also useful in the methods of the invention.Preferably, the sequence identity is at least 60%, more preferably thesequence identity is at least 70%, most preferably the sequence identityis at least 80% or 90 or 95 or 99%, or any integer from 60-100% sequenceidentity in ascending order.

[0082] In addition, it is preferred that the polypeptide has at least10% of the activity of a fungal histidinol-phosphatase. More preferably,the polypeptide has at least 25%, at least 50%, at least 75% or at least90% of the activity of a fungal histidinol-phosphatase. Most preferably,the polypeptide has at least 10%, at least 25%, at least 50%, at least75% or at least 90% of the activity of the M. griseahistidinol-phosphatase protein.

[0083] Thus, in another embodiment, the invention provides a method foridentifying a test compound as a candidate for a fungicide, comprising:

[0084] a) contacting a test compound with at least one polypeptideselected from the group consisting of: a polypeptide having at least tenconsecutive amino acids of a fungal histidinol-phosphatase; apolypeptide having at least 50% sequence identity with a fungalhistidinol-phosphatase; and a polypeptide having at least 10% of theactivity of a fungal histidinol-phosphatase; and

[0085] b) detecting the presence and/or absence of binding between saidtest compound and said polypeptide;

[0086] wherein binding indicates that said test compound is a candidatefor an antibiotic.

[0087] Any technique for detecting the binding of a ligand to its targetmay be used in the methods of the invention. For example, the ligand andtarget are combined in a buffer. Many methods for detecting the bindingof a ligand to its target are known in the art, and include, but are notlimited to the detection of an immobilized ligand-target complex or thedetection of a change in the properties of a target when it is bound toa ligand. For example, in one embodiment, an array of immobilizedcandidate ligands is provided. The immobilized ligands are contactedwith a histidinol-phosphatase protein or a fragment or variant thereof,the unbound protein is removed and the bound histidinol-phosphatase isdetected. In a preferred embodiment, bound histidinol-phosphatase isdetected using a labeled binding partner, such as a labeled antibody. Ina variation of this assay, histidinol-phosphatase is labeled prior tocontacting the immobilized candidate ligands. Preferred labels includefluorescent or radioactive moieties. Preferred detection methods includefluorescence correlation spectroscopy (FCS) and FCS-related confocalnanofluorimetric methods.

[0088] Once a compound is identified as a candidate for an antibiotic,it can be tested for the ability to inhibit histidinol-phosphataseenzymatic activity. The compounds can be tested using either in vitro orcell based assays. Alternatively, a compound can be tested by applyingit directly to a fungus or fungal cell, or expressing it therein, andmonitoring the fungus or fungal cell for changes or decreases in growth,development, viability, pathogenicity, or alterations in geneexpression. Thus, in one embodiment, the invention provides a method fordetermining whether a compound identified as an antibiotic candidate byan above method has antifungal activity, further comprising: contactinga fungus or fungal cells with said antifungal candidate and detecting adecrease in the growth, viability, or pathogenicity of said fungus orfungal cells.

[0089] By decrease in growth, is meant that the antifungal candidatecauses at least a 10% decrease in the growth of the fungus or fungalcells, as compared to the growth of the fungus or fungal cells in theabsence of the antifungal candidate. By a decrease in viability is meantthat at least 20% of the fungal cells, or portion of the funguscontacted with the antifungal candidate are nonviable. Preferably, thegrowth or viability will be decreased by at least 40%. More preferably,the growth or viability will be decreased by at least 50%, 75% or atleast 90% or more. Methods for measuring fungal growth and cellviability are known to those skilled in the art. By decrease inpathogenicity, is meant that the antifungal candidate causes at least a10% decrease in the disease caused by contact of the fungal pathogenwith its host, as compared to the disease caused in the absence of theantifungal candidate. Preferably, the disease will be decreased by atleast 40%. More preferably, the disease will be decreased by at least50%, 75% or at least 90% or more. Methods for measuring fungal diseaseare well known to those skilled in the art, and include such metrics aslesion formation, lesion size, sporulation, respiratory failure, and/ordeath.

[0090] The ability of a compound to inhibit histidinol-phosphataseactivity can be detected using in vitro enzymatic assays in which thedisappearance of a substrate or the appearance of a product is directlyor indirectly detected. Histidinol-phosphatase catalyzes theirreversible or reversible reaction L-histidinol phosphate andH₂O=L-histidinol and orthophosphate (see FIG. 1). Methods for detectionof L-histidinol phosphate, H₂O, L-histidinol, and/or orthophosphate,include spectrophotometry, mass spectroscopy, thin layer chromatography(TLC) and reverse phase HPLC.

[0091] Thus, the invention provides a method for identifying a testcompound as a candidate for an antibiotic, comprising:

[0092] a) contacting L-histidinol phosphate and H₂O with ahistidinol-phosphatase;

[0093] b) contacting L-histidinol phosphate and H₂O withhistidinol-phosphatase and said test compound; and

[0094] c) determining the change in concentration for at least one ofthe following: L-histidinol phosphate, H₂O, L-histidinol, and/ororthophosphate,

[0095] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0096] An additional method is provided by the invention for identifyinga test compound as a candidate for an antibiotic, comprising:

[0097] a) contacting L-histidinol and orthophosphate with ahistidinol-phosphatase;

[0098] b) contacting L-histidinol and orthophosphate with ahistidinol-phosphatase and a test compound; and

[0099] c) determining the change in concentration for at least one ofthe following: L-histidinol phosphate, H₂O, L-histidinol, and/ororthophosphate,

[0100] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0101] Enzymatically active fragments of a fungal histidinol-phosphataseare also useful in the methods of the invention. For example, anenzymatically active polypeptide comprising at least 100 consecutiveamino acid residues of a fungal histidinol-phosphatase may be used inthe methods of the invention. In addition, an enzymatically activepolypeptide having at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98%sequence identity with a fungal histidinol-phosphatase may be used inthe methods of the invention. Most preferably, the polypeptide has atleast 50% sequence identity with a fungal histidinol-phosphatase and atleast 10%, 25%, 75% or at least 90% of the activity thereof.

[0102] Thus, the invention provides a method for identifying a testcompound as a candidate for an antibiotic, comprising:

[0103] a) contacting L-histidinol phosphate and H₂O with a polypeptideselected from the group consisting of: a polypeptide having at least 50%sequence identity with a histidinol-phosphatase; a polypeptide having atleast 50% sequence identity with a histidinol-phosphatase and having atleast 10% of the activity thereof; and a polypeptide comprising at least100 consecutive amino acids of a histidinol-phosphatase;

[0104] b) contacting L-histidinol phosphate and H₂O with saidpolypeptide and a test compound; and

[0105] c) determining the change in concentration for at least one ofthe following: L-histidinol phosphate, H₂O, L-histidinol, and/ororthophosphate.

[0106] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0107] An additional method is provided by the invention for identifyinga test compound as a candidate for an antibiotic, comprising:

[0108] a) contacting L-histidinol and orthophosphate with a polypeptideselected from the group consisting of: a polypeptide having at least 50%sequence identity with a histidinol-phosphatase; a polypeptide having atleast 50% sequence identity with a histidinol-phosphatase and at least10% of the activity thereof; and a polypeptide comprising at least 100consecutive amino acids of a histidinol-phosphatase;

[0109] b) contacting L-histidinol and orthophosphate, with saidpolypeptide and a test compound; and

[0110] c) determining the change in concentration for at least one ofthe following, L-histidinol phosphate, H₂O, L-histidinol, and/ororthophosphate;

[0111] wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

[0112] For the in vitro enzymatic assays, histidinol-phosphatase proteinand derivatives thereof may be purified from a fungus or may berecombinantly produced in and purified from an archael, bacterial,fungal, or other eukaryotic cell culture. Preferably these proteins areproduced using an E. coli, yeast, or filamentous fungal expressionsystem. Methods for the purification of histidinol-phosphatase may bedescribed in Millay and Houston (1973) Biochemistry 12: 2591-2596 (PMID:4351203). Other methods for the purification of histidinol-phosphataseproteins and polypeptides are known to those skilled in the art.

[0113] As an alternative to in vitro assays, the invention also providescell based assays. In one embodiment, the invention provides a methodfor identifying a test compound as a candidate for an antibiotic,comprising:

[0114] a) measuring the expression of a histidinol-phosphatase in acell, cells, tissue, or an organism in the absence of a test compound;

[0115] b) contacting said cell, cells, tissue, or organism with saidtest compound and measuring the expression of saidhistidinol-phosphatase in said cell, cells, tissue, or organism; and

[0116] c) comparing the expression of histidinol-phosphatase in steps(a) and (b);

[0117] wherein a lower expression in the presence of said test compoundindicates that said compound is a candidate for an antibiotic.

[0118] Expression of histidinol-phosphatase can be measured by detectingthe HISP1 primary transcript or mRNA, histidinol-phosphatasepolypeptide, or histidinol-phosphatase enzymatic activity. Methods fordetecting the expression of RNA and proteins are known to those skilledin the art. See, for example, Current Protocols in Molecular BiologyAusubel et al., eds., Greene Publishing and Wiley-Interscience, NewYork, 1995. The method of detection is not critical to the invention.Methods for detecting HISP1 RNA include, but are not limited toamplification assays such as quantitative reverse transcriptase-PCR,and/or hybridization assays such as Northern analysis, dot blots, slotblots, in-situ hybridization, transcriptional fusions using a HISP1promoter fused to a reporter gene, DNA assays, and microarray assays.

[0119] Methods for detecting protein expression include, but are notlimited to, immunodetection methods such as Western blots, ELISA assays,polyacrylamide gel electrophoresis, mass spectroscopy, and enzymaticassays. Also, any reporter gene system may be used to detect HISP1protein expression. For detection using gene reporter systems, apolynucleotide encoding a reporter protein is fused in frame with HISP1,so as to produce a chimeric polypeptide. Methods for using reportersystems are known to those skilled in the art.

[0120] Chemicals, compounds or compositions identified by the abovemethods as modulators, preferably inhibitors, of HISP1 expression oractivity can then be used to control fungal growth. Diseases such asrusts, mildews, and blights spread rapidly once established. Fungicidesare thus routinely applied to growing and stored crops as a preventivemeasure, generally as foliar sprays or seed dressings. For example,compounds that inhibit fungal growth can be applied to a fungus orexpressed in a fungus, in order to prevent fungal growth. Thus, theinvention provides a method for inhibiting fungal growth, comprisingcontacting a fungus with a compound identified by the methods of theinvention as having antifungal activity.

[0121] Antifungals and antifungal inhibitor candidates identified by themethods of the invention can be used to control -the growth of undesiredfungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota,and lichens.

[0122] Examples of undesired fungi include, but are not limited toPowdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea),White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum),Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot(Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), HoneyFungus (Armillaria gallica), Root rot (Armillaria luteobubalina),Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus(Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena),Apple-rotting Fungus (Penicillium expansum), Clubroot Disease(Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Rootpathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomycesgraminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromycesappendiculatus), Northern Leaf Spot (Cochliobolus carbonum), MiloDisease (Periconia circinata), Southern Corn Blight (Cochliobolusheterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe(Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), WheatHead Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusariumculmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black StemRust (Puccinia graminis), White mold (Sclerotinia sclerotiorum),diseases of animals such as infections of lungs, blood, brain, skin,scalp, nails or other tissues (Aspergillus fumigatus Aspergillus sp.Fusraium sp., Trichophyton sp., Epidermophyton sp., and Microsporum sp.,and the like).

[0123] Also provided is a method of screening for an antibiotic bydetermining whether a test compound is active against the geneidentified (SEQ ID NO: 1 or SEQ ID NO: 2), its gene product (SEQ ID NO:3), or the biochemical pathway or pathways it functions on.

[0124] In one particular embodiment, the method is performed byproviding an organism having a first form of the gene corresponding toeither SEQ ID NO: 1 or SEQ ID NO: 2, either a normal form, a mutantform, a homologue, or a heterologous HISP1 gene that performs a similarfunction as HISP1. The first form of HISP1 may or may not confer agrowth conditional phenotype, i.e., a L-histidine requiring phenotype,and/or a hypersensitivity or hyposensitivity phenotype on the organismhaving that altered form. In one particular embodiment a mutant formcontains a transposon insertion. A comparison organism having a secondform of a HISP1, different from the first form of the gene is alsoprovided, and the two organisms are separately contacted with a testcompound. The growth of the two organisms in the presence of the testcompound is then compared.

[0125] Thus, in one embodiment, the invention provides a method foridentifying a test compound as a candidate for an antibiotic,comprising:

[0126] a) providing cells having one form of a histidinol-phosphatasegene, and providing comparison cells having a different form of ahistidinol-phosphatase gene; and

[0127] b) contacting said cells and said comparison cells with a testcompound and determining the growth of said cells and said comparisoncells in the presence of the test compound,

[0128] wherein a difference in growth between said cells and saidcomparison cells in the presence of said test compound indicates thatsaid test compound is a candidate for an antibiotic.

[0129] It is recognized in the art that the optional determination ofthe growth of said first organism and said comparison second organism inthe absence of any test compounds may be performed to control for anyinherent differences in growth as a result of the different genes. It isalso recognized that any combination of two different forms of a HISP1gene, including normal genes, mutant genes, homologues, and functionalhomologues may be used in this method. Growth and/or proliferation of anorganism is measured by methods well known in the art such as opticaldensity measurements, and the like. In a preferred embodiment theorganism is Magnaporthe grisea.

[0130] Conditional lethal mutants may identify particular biochemicaland/or genetic pathways given that at least one identified target geneis present in that pathway. Knowledge of these pathways allows for thescreening of test compounds as candidates for antibiotics as inhibitorsof the substrates, products and enzymes of the pathway. Pathways knownin the art may be found at the Kyoto Encyclopedia of Genes and Genomesand in standard biochemistry texts (Lehninger, A., D. Nelson, et al.(1993) Principles of Biochemistry. New York, Worth Publishers).

[0131] Thus, in one embodiment, the invention provides a method forscreening for test compounds acting against the biochemical and/orgenetic pathway or pathways in which HISP1 functions, comprising:

[0132] a) providing cells having one form of a gene in the L-histidinebiochemical and/or genetic pathway and providing comparison cells havinga different form of said gene.

[0133] b) contacting said cells and said comparison cells with a testcompound; and

[0134] c) determining the growth of said cells and said comparison cellsin the presence of said test compound;

[0135] wherein a difference in growth between said cells and saidcomparison cells in the presence of said test compound indicates thatsaid test compound is a candidate for an antibiotic.

[0136] The use of multi-well plates for screening is a format thatreadily accommodates multiple different assays to characterize variouscompounds, concentrations of compounds, and fungal strains in varyingcombinations and formats. Certain testing parameters for the screeningmethod can significantly affect the identification of growth inhibitors,and thus can be manipulated to optimize screening efficiency and/orreliability. Notable among these factors are variable sensitivities ofdifferent mutants, increasing hypersensitivity with increasingly lesspermissive conditions, an apparent increase in hypersensitivity withincreasing compound concentration, and other factors known to those inthe art.

[0137] Conditional lethal mutants may identify particular biochemicaland/or genetic pathways given that at least one identified target geneis present in that pathway. Knowledge of these pathways allows for thescreening of test compounds as candidates for antibiotics. Pathwaysknown in the art may be found at the Kyoto Encyclopedia of Genes andGenomes and in standard biochemistry texts (Lehninger, A., D. Nelson, etal. (1993) Principles of Biochemistry. New York, Worth Publishers).

[0138] Thus, in one embodiment, the invention provides a method forscreening for test compounds acting against the biochemical and/orgenetic pathway or pathways in which HISP1 functions, comprising:

[0139] (a) providing paired growth media comprising a first medium and asecond medium, wherein said second medium contains a higher level ofL-histidine than said first medium;

[0140] b) contacting an organism with a test compound;

[0141] (c) inoculating said first and said second media with saidorganism; and

[0142] d) determining the growth of said organism;

[0143] wherein a difference in growth of the organism between said firstand said second media indicates that said test compound is a candidatefor an antibiotic.

[0144] It is recognized in the art that determination of the growth ofsaid organism in the paired media in the absence of any test compoundsmay be performed to control for any inherent differences in growth as aresult of the different media. Growth and/or proliferation of anorganism is measured by methods well known in the art such as opticaldensity measurements, and the like. In a preferred embodiment, theorganism is Magnaporthe grisea.

Experimental EXAMPLE 1 Construction of Plasmids with a TransposonContaining a Selectable Marker

[0145] Construction of Sif transposon: Sif was constructed using theGPS3 vector from the GPS-M mutagenesis system from New England Biolabs,Inc. (Beverly, Mass.) as a backbone. This system is based on thebacterial transposon Tn7. The following manipulations were done to GPS3according to Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press. The kanamycin resistancegene (npt) contained between the Tn7 arms was removed by EcoRVdigestion. The bacterial hygromycin B phosphotransferase (hph) gene(Gritz and Davies (1983) Gene 25: 179-88 (PMID: 6319235)) under controlof the Aspergillus nidulans trpC promoter and terminator (Mullaney etal. (1985) Mol Gen Genet 199: 37-45 (PMID: 3158796)) was cloned by aHpaI/EcoRV blunt ligation into the Tn7 arms of the GPS3 vector yieldingpSif1. Excision of the ampicillin resistance gene (bla) from pSif1 wasachieved by cutting pSif1 with XmnI and BglI followed by a T4 DNApolymerase treatment to remove the 3′ overhangs left by the BglIdigestion and relegation of the plasmid to yield pSif. Top 10F′electrocompetent E. coli cells (Invitrogen) were transformed withligation mixture according to manufacturer's recommendations.Transformants containing the Sif transposon were selected on LB agar(Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual, ColdSpring Harbor Laboratory Press.) containing 50 ug/ml of hygromycin B(Sigma Chem. Co., St. Louis, Mo.).

EXAMPLE 2 Construction of a Fungal Cosmid Library

[0146] Cosmid libraries were constructed in the pcosKA5 vector (Hamer etal. (2001) Proc Natl Acad Sci USA 98: 5110-15 (PMID: 11296265)) asdescribed in Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press. Cosmid libraries werequality checked by pulsed-field gel electrophoresis, restrictiondigestion analysis, and PCR identification of single genes.

EXAMPLE 3 Construction of Cosmids with Transposon Insertion into FungalGenes

[0147] Sif Transposition into a Cosmid: Transposition of Sif into thecosmid framework was carried out as described by the GPS-M mutagenesissystem (New England Biolabs, Inc.). Briefly, 2 ul of the 10×GPS buffer,70 ng of supercoiled pSIF, 8-12 μg of target cosmid DNA were mixed andtaken to a final volume of 20 ul with water. 1 ul of transposase(TnsABC) was added to the reaction and incubated for 10 minutes at 37°C. to allow the assembly reaction to happen. After the assembly reaction1 ul of start solution was added to the tube, mixed well and incubatedfor 1 hour at 37° C. followed by heat inactivation of the proteins at75° C. for 10 min. Destruction of the remaining untransposed pSif wasdone by PISceI digestion at 37° C. for 2 hours followed by 10 minincubation at 75° C. to inactivate the proteins. Transformation ofTop10F′ electrocompetent cells (Invitrogen) was done according tomanufacturers recommendations. Sif-containing cosmid transformants wereselected by growth on LB agar plates containing 50 ug/ml of hygromycin B(Sigma Chem. Co.) and 100 ug/ml of Ampicillin (Sigma Chem. Co.).

EXAMPLE 4 High Throughput Preparation and Verification of TransposonInsertion into the M. grisea HISP1 Gene

[0148]E. coli strains containing cosmids with transposon insertions werepicked to 96 well growth blocks (Beckman Co.) containing 1.5 ml of TB(Terrific Broth, Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press) supplemented with 50 ug/mlof ampicillin. Blocks were incubated with shaking at 37 C overnight. E.coli cells were pelleted by centrifugation and cosmids were isolated bya modified alkaline lysis method (Narra et al. (1997) Genome Res 7:1072-84 (PMID: 9371743)). DNA quality was checked by electrophoresis onagarose gels. Cosmids were sequenced using primers from the ends of eachtransposon and commercial dideoxy sequencing kits (Big Dye Terminators,Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNAsequencer (Perkin Elmer Co.).

[0149] DNA sequences adjacent to the site of the insertion werecollected and used to search DNA and protein databases using the BLASTalgorithms (Altschul et al. (1997) Nucleic Acids Res 25: 3389-3402(PMID: 9254694)). A single insertion of SIF into the Magnaporthe griseaHISP 1 gene was chosen for further analysis. This construct wasdesignated cpgmra0012021b05 and it contains the SIF transposonapproximately 100 nucleotides before the start codon relative to theSchizosaccharomyces pombe homologue (total length: 315 amino acids,GENBANK: 3183028, SWISS-PROT: O14059), and is predicted to eliminate orreduce gene function.

EXAMPLE 5 Preparation of HISP1 Cosmid DNA and Transformation ofMagnaporthe grisea

[0150] Cosmid DNA from the HISP1 transposon tagged cosmid clone wasprepared using QIAGEN Plasmid Maxi Kit (QIAGEN), and digested by PI-PspI(New England Biolabs, Inc.). Fungal electro-transformation was performedessentially as described (Wu et al. (1997) MPMI 10: 700-708). Briefly,M. grisea strain Guy 11 was grown in complete liquid media (Talbot etal. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) shaking at 120 rpmfor 3 days at 25° C. in the dark. Mycelia was harvested and washed withsterile H₂O and digested with 4 mg/ml beta-glucanase (InterSpex) for 4-6hours to generate protoplasts. Protoplasts were collected bycentrifugation and resuspended in 20% sucrose at the concentration of2×10⁸ protoplasts/ml. 50 ul protoplast suspension was mixed with 10-20ug of the cosmid DNA and pulsed using Gene Pulser II (BioRad) set withthe following parameters: resistance 200 ohm, capacitance 25 uF, voltage0.6 kV. Transformed protoplasts were regenerated in complete agar media(C M, Talbot et al (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) withthe addition of 20% sucrose for one day, then overlayed with CM agarmedia containing hygromycin B (250 ug/ml) to select transform ants.Transformants were screened for homologous recombination events in thetarget gene by PCR (Hamer et al. (2001) Proc Natl Acad Sci USA 98:5110-15 (PMID: 11296265)). Two independent strains were identified andare hereby referred to as KO1-1 and KO1-3, respectively.

EXAMPLE 6 Effect of Transposon Insertion on Magnaporthe Pathogenicity

[0151] The target fungal strains, KO1-1 and KO1-3, obtained in Example 5and the wild type strain, Guy11, were subjected to a pathogenicity assayto observe infection over a 1-week period. Rice infection assays wereperformed using Indian rice cultivar C039 essentially as described inValent et al. ((1991) Genetics 127: 87-101 (PMID: 2016048)). All threestrains were grown for spore production on complete agar media. Sporeswere harvested and the concentration of spores adjusted for whole plantinoculations. Two-week-old seedlings of cultivar CO39 were sprayed with12 ml of conidial suspension (5×10⁴ conidia per ml in 0.01% Tween-20(Polyoxyethylensorbitan monolaureate) solution). The inoculated plantswere incubated in a dew chamber at 27° C. in the dark for 36 hours, andtransferred to a growth chamber (27° C. 12 hours/21° C. 12 hours 70%humidity) for an additional 5.5 days. Leaf samples were taken at 3, 5,and 7 days post-inoculation and examined for signs of successfulinfection (i.e. lesions). FIG. 2 shows the effects of HISPI genedisruption on Magnaporthe infection at five days post-inoculation.

EXAMPLE 7 Verification of HISP1 Gene Function by Analysis of NutritionalRequirements

[0152] The fungal strains, KO1-1 and KO1-3, containing the HISP1disrupted gene obtained in Example 5 were analyzed for their nutritionalrequirement for histidine using the PM5 phenotype microarray fromBiolog, Inc. (Hayward, Calif.). The PM5 plate tests for the auxotrophicrequirement for 94 different metabolites. The inoculating fluid consistsof 0.05% Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5 mM NaNO₃, 6.7 mMKCl, 3.5 mM Na₂SO₄, 11 mM KH₂PO₄, 0.01%p-iodonitrotetrazolium violet,0.1 mM MgCl₂, 1.0 mM CaCl₂ and trace elements. Final concentrations oftrace elements are: 7.6 μM ZnCl₂, 2.5 μM MnCl₂.4H₂O, 1.8 μM FeCl₂.4H₂O,0.71 μM CoCl₂.6H₂O, 0.64 μM CuCl₂.2H₂O, 0.62 μM Na₂MoO₄, 18 μM H₃BO₃. pHadjusted to 6.0 with NaOH. Spores for each strain were harvested intothe inoculating fluid. The spore concentrations were adjusted to 2×10⁵spores/ml. 100 μl of spore suspension were deposited into each well ofthe microtiter plates. The plates were incubated at 25° C. for 7 days.Optical density (OD) measurements at 490 nm and 750 nm were taken daily.The OD₄₉₀ measures the extent of tetrazolium dye reduction and the levelof growth, and OD₇₅₀ measures growth only. Turbidity=OD₄₉₀+OD₇₅₀. Dataconfirming the annotated gene function is presented as a graph ofTurbidity vs. Time showing both the mutant fungi and the wild-typecontrol in the absence (FIG. 3A) and presence (FIG. 3B) of L-histidine.

EXAMPLE 8 Cloning and Expression Strategies, Extraction and Purificationof Histidinol-Phosphatase Protein.

[0153] The following protocol may be employed to obtain a purifiedhistidinol-phosphatase protein.

[0154] Cloning and Expression Strategies:

[0155] A HISP1 cDNA gene can be cloned into E. coli (pETvectors-Novagen), Baculovirus (Pharmingen) and Yeast (Invitrogen)expression vectors containing His/fusion protein tags and the expressionof recombinant protein can be evaluated by SDS-PAGE and Western blotanalysis.

[0156] Extraction:

[0157] Extract recombinant protein from 250 ml cell pellet in 3 ml ofextraction buffer by sonicating 6 times, with 6 sec pulses at 4° C.Centrifuge extract at 15000×g for 10 min and collect supernatant. Assessbiological activity of the recombinant protein by activity assay.

[0158] Purification:

[0159] Purify recombinant protein by Ni-NTA affinity chromatography(Qiagen). Purification protocol: perform all steps at 4° C.:

[0160] Use 3 ml Ni-beads (Qiagen)

[0161] Equilibrate column with the buffer

[0162] Load protein extract

[0163] Wash with the equilibration buffer

[0164] Elute bound protein with 0.5 M imidazole

EXAMPLE 9 Assays for Testing Binding of Test Compounds toHistidinol-Phosphatase

[0165] The following protocol may be employed to identify test compoundsthat bind to the histidinol-phosphatase protein.

[0166] Purified full-length histidinol-phosphatase polypeptide with aHis/fusion protein tag (Example 8) is bound to a HisGrab™ Nickel CoatedPlate (Pierce, Rockford, Ill.) following manufacturer's instructions.

[0167] Buffer conditions are optimized (e.g. ionic strength or pH,Millay and Houston (1973) Biochemistry 12: 2591-2596 (PMID: 4351203))for binding of radiolabeled L-Histidinol phosphate (custom made,PerkinElmer Life Sciences, Inc., Boston, Mass.) to the boundhistidinol-phosphatase.

[0168] Screening of test compounds is performed by adding test compoundand L- Histidinol phosphate (custom made, PerkinElmer Life Sciences,Inc., Boston, Mass.) to the wells of the HisGrab™ plate containing boundhistidinol-phosphatase.

[0169] The wells are washed to remove excess labeled ligand andscintillation fluid (Scintiverse®, Fisher Scientific) is added to eachwell.

[0170] The plates are read in a microplate scintillation counter.

[0171] Candidate compounds are identified as wells with lowerradioactivity as compared to control wells with no test compound added.

[0172] Additionally, a purified polypeptide comprising 10-50 amino acidsfrom the M. grisea histidinol-phosphatase is screened in the same way. Apolypeptide comprising 10-50 amino acids is generated by subcloning aportion of the HISP1 gene into a protein expression vector that adds aHis-Tag when expressed (see Example 8). Oligonucleotide primers aredesigned to amplify a portion of the HISP1 gene using the polymerasechain reaction amplification method. The DNA fragment encoding apolypeptide of 10-50 amino acids is cloned into an expression vector,expressed in a host organism and purified as described in Example 8above.

[0173] Test compounds that bind HISPI are further tested for antibioticactivity. M. grisea is grown as described for spore production onoatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID:8312740)). Spores are harvested into minimal media (Talbot et al. (1993)Plant Cell 5: 1575-1590 (PMID: 8312740)) to a concentration of 2×10⁵spores/ml and the culture is divided. The test compound is added to oneculture to a final concentration of 20-100 μg/ml. Solvent only is addedto the second culture. The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. The growthcurves of the solvent control sample and. the test compound sample arecompared. A test compound is an antibiotic candidate if the growth ofthe culture containing the test compound is less than the growth of thecontrol culture.

EXAMPLE 10 Assays for Testing Inhibitors or Candidates for Inhibition ofHistidinol-Phosphatase Activity

[0174] The enzymatic activity of histidinol-phosphatase is determined inthe presence and. absence of candidate compounds in a suitable reactionmixture, such as described by Millay and Houston (1973) Biochemistry 12:2591-2596 (PMID: 4351203). Candidate compounds are identified when adecrease in products or a lack of decrease in substrates is detectedwith the reaction proceeding in either direction.

[0175] Additionally, the enzymatic activity of a polypeptide comprising10-50 amino acids from the M. grisea histidinol-phosphatase isdetermined in the presence and absence of candidate compounds in asuitable reaction mixture, such as described by Millay and Houston(1973) Biochemistry 12: 2591-2596 (PMID: 4351203). A polypeptidecomprising 10-50 amino acids is generated by subcloning a portion of theHISP1 gene into a protein expression vector that adds a His-Tag whenexpressed (see Example 8). Oligonucleotide primers are designed toamplify a portion of the HISP1 gene using polymerase chain reactionamplification method. The DNA fragment encoding a polypeptide of 10-50amino acids is cloned into an expression vector, expressed and purifiedas described in Example 8 above.

[0176] Test compounds identified as inhibitors of HISP1 activity arefurther tested for antibiotic activity. Magnaporthe grisea fungal cellsare grown under standard fungal growth conditions that are well knownand described in the art. M. grisea is grown as described for sporeproduction on oatmeal agar media (Talbot et al. (1993) Plant Cell 5:1575-1590 (PMID: 8312740)). Spores are harvested into minimal media(Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) to aconcentration of 2×10⁵ spores/ml and the culture is divided. The testcompound is added to one culture to a final concentration of 20-100μg/ml. Solvent only is added to the second culture. The plates areincubated at 25° C. for seven days and optical density measurements at590 nm are taken daily. The growth curves of the solvent control sampleand the test compound sample are compared. A test compound is anantibiotic candidate if the growth of the culture containing the testcompound is less than the growth of the control culture.

EXAMPLE 11 Assays for Testing Compounds for Alteration ofHistidinol-phosphatase Gene Expression

[0177]Magnaporthe grisea fungal cells are grown under standard fungalgrowth conditions that are well known and described in the art.Wild-type M. grisea spores are harvested from cultures grown on completeagar or oatmeal agar media after growth for 10-13 days in the light at25° C. using a moistened cotton swab. The concentration of spores isdetermined using a hemacytometer and spore suspensions are prepared in aminimal growth medium to a concentration of 2×10⁵ spores per ml. 25 mlcultures are prepared to which test compounds will be added at variousconcentrations. A culture with no test compound present is included as acontrol. The cultures are incubated at 25° C. for 3 days after whichtest compound or solvent only control is added. The cultures areincubated an additional 18 hours. Fungal mycelia is harvested byfiltration through Miracloth (CalBiochem®, La Jolla, Calif.), washedwith water and frozen in liquid nitrogen. Total RNA is extracted withTRIZOL® Reagent using the methods provided by the manufacturer (LifeTechnologies, Rockville, Md.). Expression is analyzed by Northernanalysis of the RNA samples as described (Sambrook et al. (1989)Molecular Cloning, a Laboratory Manual, Cold Spring Harbor LaboratoryPress) using a radiolabeled fragment of the HISP1 gene as a probe. Testcompounds resulting in a reduced level of HISP1 mRNA relative to theuntreated control sample are identified as candidate antibioticcompounds.

EXAMPLE 12 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of Histidinol-Phosphatase with NoActivity

[0178]Magnaporthe grisea fungal cells containing a mutant form of theHISP1 gene which abolishes enzyme activity, such as a gene containing atransposon insertion (see Examples 4 and 5), are grown under standardfungal growth conditions that are well known and described in the art.Magnaporthe grisea spores are harvested from cultures grown on completeagar medium containing 4 mM L-histidine (Sigma-Aldrich Co.) after growthfor 10-13 days in the light at 25° C. using a moistened cotton swab. Theconcentration of spores is determined using a hemacytometer and sporesuspensions are prepared in a minimal growth medium containing 100 μML-histidine to a concentration of 2×10⁵ spores per ml. Approximately4×10⁴ spores are added to each well of 96-well plates to which a testcompound is added (at varying concentrations). The total volume in eachwell is 200 μl. Wells with no test compound present (growth control),and wells without cells are included as controls (negative control). Theplates are incubated at 25° C. for seven days and optical densitymeasurements at 590 nm are taken daily. Wild type cells are screenedunder the same conditions. The effect of each compound on the mutantand. wild-type fungal strains is measured against the growth control andthe percent of inhibition is calculated as the OD₅₉₀ (fungal strain plustest compound)/ OD₅₉₀ (growth control)×100. The percent of growthinhibition as a result of a test compound on a fungal strain and that onthe wild type cells are compared. Compounds that show differentialgrowth inhibition between the mutant and the wild type are identified aspotential antifungal compounds. Similar protocols may be found in Kirschand DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

EXAMPLE 13 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of Histidinol-Phosphatase with ReducedActivity

[0179]Magnaporthe grisea fungal cells containing a mutant form of theHISP1 gene, such as a promoter truncation that reduces expression, aregrown under standard fungal growth conditions that are well known anddescribed in the art. A promoter truncation is made by deleting aportion of the promoter upstream of the transcription start site usingstandard molecular biology techniques that are well known and describedin the art (Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea sporesare harvested from cultures grown on complete agar medium containing 4mM L-histidine (Sigma-Aldrich Co.) after growth for 10-13 days in thelight at 25° C. using a moistened cotton swab. The concentration ofspores is determined using a hemacytometer and spore suspensions areprepared in a minimal growth medium to a concentration of 2×10⁵ sporesper ml. Approximately 4×10⁴ spores are added to each well of 96-wellplates to which a test compound is added (at varying concentrations).The total volume in each well is 200 μl. Wells with no test compoundpresent (growth control), and wells without cells are included ascontrols (negative control). The plates are incubated at 25° C. forseven days and optical density measurements at 590 nm are taken daily.Wild type cells are screened under the same conditions. The effect ofeach compound on the mutant and wild-type fungal strains is measuredagainst the growth control and the percent of inhibition is calculatedas the OD₅₉₀ (fungal strain plus test compound)/ OD₅₉₀ (growthcontrol)×100. The percent of growth inhibition as a result of a testcompound on a fungal strain and that on the wild-type cells arecompared. Compounds that show differential growth inhibition between themutant and the wild type are identified as potential antifungalcompounds. Similar protocols may be found in Kirsch and DiDomenico((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

EXAMPLE 14 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of a L-Histidine Biosynthetic Gene withNo Activity

[0180]Magnaporthe grisea fungal cells containing a mutant form of a genein the L-histidine biosynthetic pathway (e.g. Histidinol dehydrogenase(E.C. 1.1.1.23)) are grown under standard fungal growth conditions thatare well known and described in the art. Magnaporthe grisea spores areharvested from cultures grown on complete agar medium containing 4 mML-histidine (Sigma-Aldrich Co.) after growth for 10-13 days in the lightat 25° C. using a moistened cotton swab. The concentration of spores isdetermined using a hemacytometer and spore suspensions are prepared in aminimal growth medium containing 100 μM L-histidine to a concentrationof 2×11⁵ spores per ml. Approximately 4×10⁴ spores or cells areharvested and added to each well of 96-well plates to which growth mediais added in addition to an amount of test compound (at varyingconcentrations). The total volume in each well is 200 μl. Wells with notest compound present, and wells without cells are included as controls.The plates are incubated at 25° C. for seven days and optical densitymeasurements at 590 nm are taken daily. Wild type cells are screenedunder the same conditions. The effect of each compound on the mutant andwild-type fungal strains is measured against the growth control and thepercent of inhibition is calculated as the OD₅₉₀ (fungal strain plustest compound)/OD₅₉₀ (growth control)×100. The percent of growthinhibition as a result of a test compound on a fungal strain and that onthe wild type cells are compared. Compounds that show differentialgrowth inhibition between the mutant and the wild-type are identified aspotential antifungal compounds. Similar protocols may be found in Kirschand DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

EXAMPLE 15 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of a L-Histidine Biosynthetic Gene withReduced Activity

[0181]Magnaporthe grisea fungal cells containing a mutant form of a genein the L-histidine biosynthetic pathway (e.g. Histidinol dehydrogenase(E.C. 1.1.1.23)), such as a promoter truncation that reduces expression,are grown under standard fungal growth conditions that are well knownand described in the art. A promoter truncation is made by deleting aportion of the promoter upstream of the transcription start site usingstandard molecular biology techniques that are well known and describedin the art (Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea fungalcells containing a mutant form of are grown under standard fungal growthconditions that are well known and described in the art. Magnaporthegrisea spores are harvested from cultures grown on complete agar mediumcontaining 4 mM L-histidine (Sigma-Aldrich Co.) after growth for 10-13days in the light at 25° C. using a moistened cotton swab. Theconcentration of spores is determined using a hemacytometer and sporesuspensions are prepared in a minimal growth medium to a concentrationof 2×10⁵ spores per ml. Approximately 4×10⁴ spores or cells areharvested and added to each well of 96-well plates to which growth mediais added in addition to an amount of test compound (at varyingconcentrations). The total volume in each well is 200 μl. Wells with notest compound present, and wells without cells are included as controls.The plates are incubated at 25° C. for seven days and optical densitymeasurements at 590 nm are taken daily. Wild type cells are screenedunder the same conditions. The effect of each compound on the mutant andwild-type fungal strains is measured against the growth control and thepercent of inhibition is calculated as the OD₅₉₀ (fungal strain plustest compound)/OD₅₉₀ (growth control)×100. The percent of growthinhibition as a result of a test compound on a fungal strain and that onthe wild type cells are compared. Compounds that show differentialgrowth inhibition between the mutant and the wild type are identified aspotential antifungal compounds. Similar protocols may be found in Kirschand DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

EXAMPLE 16 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Fungal HISP1 and a Second Fungal Strain Containing aHeterologous HISP1 Gene

[0182] Wild-type Magnaporthe grisea fungal cells and M. grisea fungalcells lacking a functional HISP1 gene and containing a heterologousHISP1 gene are grown under standard fungal growth conditions that arewell known and described in the art. A M. grisea strain carrying aheterologous HISP1 gene is made as follows:

[0183] A M. grisea strain is made with a nonfunctional HISP1 gene, suchas one containing a transposon insertion in the native gene (seeExamples 4 and 5).

[0184] A construct containing a heterologous HISP1 gene is made bycloning the heterologous HISP1 gene into a fungal expression vectorcontaining a trpC promoter and terminator (e.g. pCB1003, Carroll et al.(1994) Fungal Gen News Lett 41: 22) using standard molecular biologytechniques that are well known and described in the art (Sambrook et al(1989) Molecular Cloning, a Laboratory Manual, Cold Spring HarborLaboratory Press).

[0185] The said construct is used to transform the M. grisea strainlacking a functional HISP1 gene (see Example 5). Transformants areselected on minimal agar medium lacking L-histidine. Only transformantscarrying a functional HISP1 gene will grow. Wild-type strains ofMagnaporthe grisea and strains containing a heterologous form of IISPIare grown under standard fungal growth conditions that are well knownand described in the art. Magnaporthe grisea spores are harvested fromcultures grown on complete agar medium after growth for 10-13 days inthe light at 25° C. using a moistened cotton swab. The concentration ofspores is determined using a hemacytometer and spore suspensions areprepared in a minimal growth medium to a concentration of 2×10⁵ sporesper ml. Approximately 4×10⁴ spores or cells are harvested and added toeach well of 96-well plates to which growth media is added in additionto an amount of test compound (at varying concentrations). The totalvolume in each well is 200 μl. Wells with no test compound present, andwells without cells are included as controls. The plates are incubatedat 25° C. for seven days and optical density measurements at 590 nm aretaken daily. The effect of each compound on the wild-type andheterologous fungal strains is measured against the growth control andthe percent of inhibition is calculated as the OD₅₉₀ (fungal strain plustest compound)/OD₅₉₀ (growth control)×100. The percent of growthinhibition as a result of a test compound on the wild-type andheterologous fungal strains are compared. Compounds that showdifferential growth inhibition between the wild-type and heterologousstrains are identified as potential antifungal compounds withspecificity to the native or heterologous HISP1 gene products. Similarprotocols may be found in Kirsch and DiDomenico ((1994) Biotechnology26: 177-221 (PMID: 774-9303)).

EXAMPLE 17 Pathway Specific In Vivo Assay Screening Protocol

[0186]Magnaporthe grisea fungal cells are grown under standard fungalgrowth conditions that are well known and described in the art.Wild-type M. grisea spores are harvested from cultures grown on oatmealagar media after growth for 10-13 days in the light at 25° C. using amoistened cotton swab. The concentration of spores is determined using ahemocytometer and spore suspensions are prepared in a minimal growthmedium and a minimal growth medium containing 4 mM L-histidine(Sigma-Aldrich Co.) to a concentration of 2×10⁵ spores per ml. Theminimal growth media contains carbon, nitrogen, phosphate, and sulfatesources, and magnesium, calcium, and trace elements (for example, seeinoculating fluid in Example 7). Spore suspensions are added to eachwell of a 96-well microtiter plate (approximately 4×10⁴ spores/well).For each well containing a spore suspension in minimal media, anadditional well is present containing a spore suspension in minimalmedium containing 4 mM L-histidine. Test compounds are added to wellscontaining spores in minimal media and minimal media containingL-histidine. The total volume in each well is 200 μl. Both minimal mediaand L-histidine containing media wells with no test compound areprovided as controls. The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. A compoundis identified as a candidate for an antibiotic acting against theL-histidine biosynthetic pathway when the observed growth in the wellcontaining minimal media is less than the observed growth in the wellcontaining L-histidine as a result of the addition of the test compound.Similar protocols may be found in Kirsch and DiDomenico ((1994)Biotechnology 26: 177-221 (PMID: 7749303)).

[0187] While the foregoing describes certain embodiments of theinvention, it will be understood by those skilled in the art thatvariations and modifications may be made and still fall within the scopeof the invention. The foregoing examples are intended to exemplifyvarious specific embodiments of the invention and do not limit its scopein any manner.

1 3 1 1002 DNA Magnaporthe grisea 1 atggccttca caatgcactc gcactccgggcaattctgcc cgggccacgc aaaagaccag 60 ttggaagacg tcatcctcca tgccatcagcataggataca agaccatggg tctcagtgag 120 cacatgccac ggacccaact gtgcgatctatatccagaag aacttgtgcc tgacccgcac 180 gcctccctcg cggagctgat gccgcgccacgctgcctaca tgaccgaggc gcgtcggctg 240 caaaagaagt acgccgatcg catcaccctcctcatcggct tcgagggcga gttcatccgg 300 tccgagtacg ggacactggt gcgctcgctggccgacggca acggcgaccc ttcctacttc 360 cagaacggcg acagcaagct tgtcaccgacgccggcaagg tcgactattt catcggctcg 420 ctgcaccacg gcgccggcgg catccccatcgactttgacc gcgccaccta cctacgctcc 480 gttgaggccg ccggccccaa tggcgaggaggatctatttg tgcactacta cgaccagcag 540 tttgagatgc tccaggccct gaggccacccatcgtcggcc actttgatct gatccgcctg 600 atgagcgagg agcctgggcg caatccgagcgcctggtccc cgaaccgcgt ctggccgctc 660 atcaagcgga acctcgcgtt cgttgcgagctacggcggct ggctcgagtg caactcgagt 720 gcgctccgca aggggctcgc cgaaccgtacccgtgccggc ccatcgcgga ggaatgggta 780 aggctgggcg gtaagttcac aatgtctgacgacagccacg gcatcgcgca ggttgccaca 840 aactatgtgc gagccctgga ctacctcgagtcgctcggcg tgaacgaggt ctggacgtat 900 gaccgagcta aagagggatc agagcttgtggagaagggtg tgtcgtttac agagtttcgc 960 ggttccctga gactcccaac aacggcgtccaagacatcct ga 1002 2 1717 DNA Magnaporthe grisea 2 gggcaacggg aattcatagattgcaccgaa ggcggggctt gcatcaacaa gttttcaata 60 ctggtgtggc cacctgatttcatttaccgg atcaggaagg atatcctagc aagctgccaa 120 cattaatgga gcaagtaaaacgtattcaag cctccgagga cccagcagcc gccctcgcgc 180 cccgaaatac gcgcgctttcaggcattgct tgagcctatc tttggtgggg ttacacttta 240 aaaaaagttg ttcctgcgtgcctcccatcc aatgcctggc atccatctta gctgaggcag 300 gtaccagaca cctgggtaatgaatgctaag agtcaatctc tctaggaatt taacggattt 360 gattggctag caaacattgattgatttttc ttcgtacatt tccatgtacg ccgtatagca 420 ttacaaagag taaaagcaagtttggcctca gttaccctaa atccagatac agacagacag 480 acgccctccc tatctgccttgaatggttga agttttaata gccggcatcc ctccgtttcc 540 actcctctct tcgtcatcatcaggtatcat ctgacaacct tagagtcaac aactcaattg 600 catttttttt ccatcgcaaaagcaaacaca cagtcatggc cttcacaatg cactcgcact 660 ccgggcaatt ctgcccgggccacgcaaaag accagttgga agacgtcatc ctccatgcca 720 tcagcatagg atacaagaccatgggtctca gtgagcacat gccacggacc caactgtgcg 780 atctatatcc agaagaacttgtgcctgacc cgcacgcctc cctcgcggag ctgatgccgc 840 gccacgctgc ctacatgaccgaggcgcgtc ggctgcaaaa gaagtacgcc gatcgcatca 900 ccctcctcat cggcttcgagggcgagttca tccggtccga gtacgggaca ctggtgcgct 960 cgctggccga cggcaacggcgacccttcct acttccagaa cggcgacagc aagcttgtca 1020 ccgacgccgg caaggtcgactatttcatcg gctcgctgca ccacggcgcc ggcggcatcc 1080 ccatcgactt tgaccgcgccacctacctac gctccgttga ggccgccggc cccaatggcg 1140 aggaggatct atttgtgcactactacgacc agcagtttga gatgctccag gccctgaggc 1200 cacccatcgt cggccactttgatctgatcc gcctgatgag cgaggagcct gggcgcaatc 1260 cgagcgcctg gtccccgaaccgcgtctggc cgctcatcaa gcggaacctc gcgttcgttg 1320 cgagctacgg cggctggctcgagtgcaact cgagtgcgct ccgcaagggg ctcgccgaac 1380 cgtacccgtg ccggcccatcgcggaggaat gggtaaggct gggcggtaag ttcacaatgt 1440 ctgacgacag ccacggcatcgcgcaggttg ccacaaacta tgtgcgagcc ctggactacc 1500 tcgagtcgct cggcgtgaacgaggtctgga cgtatgaccg agctaaagag ggatcagagc 1560 ttgtggagaa gggtgtgtcgtttacagagt ttcgcggttc cctgagactc ccaacaacgg 1620 cgtccaagac atcctgatgggaagtcagcg ctcctcctca taggacatga ttttctttac 1680 attcctggcc tagaactagcccctaaggct tatgaaa 1717 3 333 PRT Magnaporthe grisea 3 Met Ala Phe ThrMet His Ser His Ser Gly Gln Phe Cys Pro Gly His 1 5 10 15 Ala Lys AspGln Leu Glu Asp Val Ile Leu His Ala Ile Ser Ile Gly 20 25 30 Tyr Lys ThrMet Gly Leu Ser Glu His Met Pro Arg Thr Gln Leu Cys 35 40 45 Asp Leu TyrPro Glu Glu Leu Val Pro Asp Pro His Ala Ser Leu Ala 50 55 60 Glu Leu MetPro Arg His Ala Ala Tyr Met Thr Glu Ala Arg Arg Leu 65 70 75 80 Gln LysLys Tyr Ala Asp Arg Ile Thr Leu Leu Ile Gly Phe Glu Gly 85 90 95 Glu PheIle Arg Ser Glu Tyr Gly Thr Leu Val Arg Ser Leu Ala Asp 100 105 110 GlyAsn Gly Asp Pro Ser Tyr Phe Gln Asn Gly Asp Ser Lys Leu Val 115 120 125Thr Asp Ala Gly Lys Val Asp Tyr Phe Ile Gly Ser Leu His His Gly 130 135140 Ala Gly Gly Ile Pro Ile Asp Phe Asp Arg Ala Thr Tyr Leu Arg Ser 145150 155 160 Val Glu Ala Ala Gly Pro Asn Gly Glu Glu Asp Leu Phe Val HisTyr 165 170 175 Tyr Asp Gln Gln Phe Glu Met Leu Gln Ala Leu Arg Pro ProIle Val 180 185 190 Gly His Phe Asp Leu Ile Arg Leu Met Ser Glu Glu ProGly Arg Asn 195 200 205 Pro Ser Ala Trp Ser Pro Asn Arg Val Trp Pro LeuIle Lys Arg Asn 210 215 220 Leu Ala Phe Val Ala Ser Tyr Gly Gly Trp LeuGlu Cys Asn Ser Ser 225 230 235 240 Ala Leu Arg Lys Gly Leu Ala Glu ProTyr Pro Cys Arg Pro Ile Ala 245 250 255 Glu Glu Trp Val Arg Leu Gly GlyLys Phe Thr Met Ser Asp Asp Ser 260 265 270 His Gly Ile Ala Gln Val AlaThr Asn Tyr Val Arg Ala Leu Asp Tyr 275 280 285 Leu Glu Ser Leu Gly ValAsn Glu Val Trp Thr Tyr Asp Arg Ala Lys 290 295 300 Glu Gly Ser Glu LeuVal Glu Lys Gly Val Ser Phe Thr Glu Phe Arg 305 310 315 320 Gly Ser LeuArg Leu Pro Thr Thr Ala Ser Lys Thr Ser 325 330

What is claimed is:
 1. A method for identifying a test compound as acandidate for an antibiotic, comprising: a) contacting ahistidinol-phosphatase polypeptide with a test compound; and b)detecting the presence or absence of binding between said test compoundand said histidinol-phosphatase polypeptide, wherein binding indicatesthat said test compound is a candidate for an antibiotic.
 2. The methodof claim 1, wherein said histidinol-phosphatase polypeptide is a fungalhistidinol-phosphatase polypeptide.
 3. The method of claim 1, whereinsaid histidinol-phosphatase polypeptide is a Magnaporthehistidinol-phosphatase polypeptide.
 4. The method of claim 1, whereinsaid histidinol-phosphatase polypeptide is SEQ ID NO:
 3. 5. A method fordetermining whether the antibiotic candidate of claim 1 has antifungalactivity, further comprising: contacting a fungus or fungal cells withsaid antibiotic candidate and detecting the decrease in growth,viability, or pathogenicity of said fungus or fungal cells.
 6. A methodfor identifying a test compound as a candidate for an antibiotic,comprising: a) contacting a test compound with at least one polypeptideselected from the group consisting of: a polypeptide having at least tenconsecutive amino acids of a fungal histidinol-phosphatase; apolypeptide having at least 50% sequence identity with; and apolypeptide having at least 10% of the activity of a fungalhistidinol-phosphatase; and b) detecting the presence and/or absence ofbinding between said test compound and said polypeptide, wherein bindingindicates that said test compound is a candidate for an antibiotic.
 7. Amethod for determining whether the antibiotic candidate of claim 6 hasantifungal activity, further comprising: contacting a fungus or fungalcells with said antibiotic candidate and detecting a decrease in growth,viability, or pathogenicity of said fungus or fungal cells.
 8. A methodfor identifying a test compound as a candidate for an antibiotic,comprising: a) contacting L-histidinol phosphate and H₂O with ahistidinol-phosphatase; b) contacting L-histidinol phosphate and H₂Owith histidinol-phosphatase and a test compound; and c) determining thechange in concentration for at least one of the following: L-histidinolphosphate, H₂O, L-histidinol, and/or orthophosphate, wherein a change inconcentration for any of the above substances between steps (a) and (b)indicates that said test compound is a candidate for an antibiotic. 9.The method of claim 8, wherein said histidinol-phosphatase is a fungalhistidinol-phosphatase.
 10. The method of claim 8, wherein saidhistidinol-phosphatase is a Magnaporthe histidinol-phosphatase.
 11. Themethod of claim 8, wherein said histidinol-phosphatase is SEQ ID NO: 3.12. A method for determining whether the antibiotic candidate of claim 8has antifungal activity, further comprising: contacting a fungus orfungal cells with said antibiotic candidate and detecting a decrease ingrowth, viability, or pathogenicity of said fungus or fungal cells. 13.A method for identifying a test compound as a candidate for anantibiotic, comprising: a) contacting L-histidinol and orthophosphatewith a histidinol-phosphatase; b) contacting L-histidinol andorthophosphate with a histidinol-phosphatase and a test compound; andc)determining the change in concentration for at least one of thefollowing: L-histidinol phosphate, H₂O, L-histidinol, and/ororthophosphate, wherein a change in concentration for any of the abovesubstances between steps (a) and (b) indicates that said test compoundis a candidate for an antibiotic.
 14. The method of claim 13, whereinsaid histidinol-phosphatase is a fungal histidinol-phosphatase.
 15. Themethod of claim 13, wherein said histidinol-phosphatase is a Magnaporthehistidinol-phosphatase.
 16. The method of claim 13, wherein saidhistidinol-phosphatase is SEQ ID NO:
 3. 17. A method for determiningwhether the antibiotic candidate of claim 13 has antifungal activity,further comprising: contacting a fungus or fungal cells with saidantibiotic candidate and detecting a decrease in growth, viability, orpathogenicity of said fungus or fungal cells.
 18. A method foridentifying a test compound as a candidate for an antibiotic,comprising: a) contacting L-histidinol phosphate and H₂O with apolypeptide selected from the group consisting of: a polypeptide havingat least 50% sequence identity with histidinol-phosphatase; apolypeptide having at least 50% sequence identity with ahistidinol-phosphatase and having at least 10% of the activity thereof;and a polypeptide comprising at least 100 consecutive amino acids of ahistidinol-phosphatase; b) contacting L-histidinol phosphate and H₂Owith said polypeptide and a test compound; and c) determining the changein concentration for at least one of the following: L-histidinolphosphate, H₂O, L-histidinol, and/or orthophosphate, wherein a change inconcentration for any of the above substances between steps (a) and (b)indicates that said test compound is a candidate for an antibiotic. 19.A method for identifying a test compound as a candidate for anantibiotic, comprising: a) contacting L-histidinol and orthophosphatewith a polypeptide selected from the group consisting of: a polypeptidehaving at least 50% sequence identity with a histidinol-phosphatase; apolypeptide having at least 50% sequence identity with ahistidinol-phosphatase and at least 10% of the activity thereof; and apolypeptide comprising at least 100 consecutive amino acids of ahistidinol-phosphatase; b) contacting L-histidinol and orthophosphate,with said polypeptide and a test compound; and c) determining the changein concentration for at least one of the following: L-histidinolphosphate, H₂O, L-histidinol, and/or orthophosphate, wherein a change inconcentration for any of the above substances between steps (a) and (b)indicates that said test compound is a candidate for an antibiotic. 20.A method for identifying a test compound as a candidate for anantibiotic, comprising: a) measuring the expression of ahistidinol-phosphatase in a cell, cells, tissue, or an organism in theabsence of a test compound; b) contacting said cell, cells, tissue, ororganism with said test compound and measuring the expression of saidhistidinol-phosphatase in said cell, cells, tissue, or organism; and c)comparing the expression of histidinol-phosphatase in steps (a) and (b),wherein a lower expression in the presence of said test compoundindicates that said test compound is a candidate for an antibiotic. 21.The method of claim 20 wherein said cell, cells, tissue, or organism is,or is derived from a fungus.
 22. The method of claim 20 wherein saidcell, cells, tissue, or organism is, or is derived from a Magnaporthefungus or fungal cell.
 23. The method of claim 20, wherein saidhistidinol-phosphatase is SEQ ID NO:
 3. 24. The method of claim 20,wherein the expression of histidinol-phosphatase is measured bydetecting HISP1 mRNA.
 25. The method of claim 20, wherein the expressionof histidinol-phosphatase is measured by detectinghistidinol-phosphatase polypeptide.
 26. A method for identifying a testcompound as a candidate for an antibiotic, comprising: a) providingcells having one form of a histidinol-phosphatase gene, and providingcomparison cells having a different form of a histidinol-phosphatasegene; and b) contacting said cells and said comparison cells with a testcompound and determining the growth of said cells and comparison cellsin the presence of the test compound, wherein a difference in growthbetween said cells and said comparison cells in the presence of saidcompound indicates that said compound is a candidate for an antibiotic.27. The method of claim 26 wherein the cells and the comparison cellsare fungal cells.
 28. The method of claim 26 wherein the cells and thecomparison cells are Magnaporthe cells.
 29. The method of claim 26wherein said form and said comparison form of the histidinol-phosphataseare fungal histidinol-phosphatases.
 30. The method of claim 26, whereinat least one of the forms is a Magnaporthe histidinol-phosphatase. 31.The method of claim 26 wherein said form and said comparison form of thehistidinol-phosphatase are non-fungal histidinol-phosphatases.
 32. Themethod of claim 26 wherein one form of the histidinol-phosphatase is afungal histidinol-phosphatase, and the other form is a non-fungalhistidinol-phosphatase.
 33. A method for identifying a test compound asa candidate for an antibiotic, comprising: a) providing cells having oneform of a gene in the L-histidine biochemical and/or genetic pathway andproviding comparison cells having a different form of said gene. b)contacting said cells and said comparison cells with a test compound, c)determining the growth of said cells and said comparison cells in thepresence of said test compound, wherein a difference in growth betweensaid cells and said comparison cells in the presence of said testcompound indicates that said test compound is a candidate for anantibiotic.
 34. The method of claim 33 wherein the cells and thecomparison cells are fungal cells.
 35. The method of claim 33 whereinthe cells and the comparison cells are Magnaporthe cells.
 36. The methodof claim 33 wherein said form and said different form of the L-histidinebiosynthesis gene are fungal L-histidine biosynthesis genes.
 37. Themethod of claim 33, wherein at least one form is a MagnaportheL-histidine biosynthesis gene.
 38. The method of claim 33 wherein saidform and said different form of the L-histidine biosynthesis genes arenon-fungal L-histidine biosynthesis genes.
 39. The method of claim 33wherein one form of the L-histidine biosynthesis gene is a fungalL-histidine biosynthesis gene, and the different form is a non-fungalL-histidine biosynthesis gene.
 40. A method for determining whether theantibiotic candidate of claim 33 has antifungal activity, furthercomprising: contacting a fungus or fungal cells with said antibioticcandidate and detecting a decrease in growth, viability, orpathogenicity of said fungus or fungal cells, wherein a decrease ingrowth, viability, or pathogenicity of said fungus or fungal cellsindicates that the antibiotic candidate has antifungal activity.
 41. Amethod for identifying a test compound as a candidate for an antibiotic,comprising: (a) providing paired growth media; comprising a first mediumand a second medium, wherein said second medium contains a higher levelof L-histidine than said first medium; (b) contacting an organism with atest compound; (c) inoculating said first and said second media withsaid organism; and (d) determining the growth of said organism, whereina difference in growth of the organism between said first and saidsecond media indicates that said test compound is a candidate for anantibiotic.
 42. The method of claim 41, wherein said organism is afungus.
 43. The method of claim 41, wherein said organism isMagnaporthe.
 44. An isolated nucleic acid comprising a nucleotidesequence that encodes a polypeptide of SEQ ID NO:
 3. 45. The nucleicacid of claim 44 comprising the nucleotide sequence of SEQ ID NO:
 1. 46.An expression cassette comprising the nucleic acid of claim
 45. 47. Theisolated nucleic acid of claim 44 comprising a nucleotide sequence withat least 50 to at least 95% sequence identity to SEQ ID NO:
 1. 48. Apolypeptide consisting essentially of the amino acid sequence of SEQ IDNO:
 3. 49. A polypeptide comprising the amino acid sequence of SEQ IDNO: 3.